The outer surface of any object can be regarded as a regular or irregular curved surface. Surfaces of precise components manufactured with modern advanced technologies are already very “flat”, and it is hence difficult to see changes of “heights” on the curved surfaces of these precise components with human eyes. However, in some scientific fields requiring extreme precision, for example, in a microscopic world magnified by an optical microscope, subtle changes can be magnified many times, thereby greatly affecting observation results and quality of images photographed by the microscope. Especially, a subtle change of heights on the curved surface of a microscope slide may cause a change of a distance between a sample on the microscope slide and the microscope lens. At low magnification, a depth of field (DOF) is large (the DOF is the distance between the nearest and farthest objects in a scene that appear acceptably sharp in an image formed in front of a microscope lens or other imagers; after being focused, clear images can be formed in front and at back of the focus; and the distance between the front and the back is called the DOF), so an effect of the DOF on imaging of the sample is limited, the distance between the sample and the lens still falls within the DOF without adjusting the distance between the adjustable lens and the microscope slide, and it is hence still capable of obtaining clear images. However, at high magnification (for example, above 100 times), since the DOF is small, a subtle height change (in micron dimension) of the curved surface of the microscope slide may cause that the distance between the sample on the microscope slide and the microscope lens exceeds the DOF, the image observed is hence blurry, and the image photographed is also blurry.
As described above, the change of height on the curved surface of the microscope slide may greatly affect the quality of image at high magnification. Since a microscope slide is usually fully covered with several samples (the samples are objects to be observed which are placed on the microscope slide; a plurality of samples may be placed on the same microscope slide; and a region of the microscope slide covered by each of the samples is defined as a surface region), a plurality of observations must be done to the samples for each microscope slide. Since the microscope lens is immobile, the samples are moved to the lens for observation under the driving of a metal rail platform movable in a transverse direction and a longitudinal direction which is used for supporting the microscope slides. In order to obtain clear image, the distance between the lens and the microscope slide must be adjusted for different samples to ensure the distance between the sample and the lens is within the DOF. At low magnification, since the DOF is large, the subtle change of height on the curved surface of the microscope slide will not affect imaging definition substantially. However, at high magnification, for example above 100 times, since the DOF is small, a micrometer-scale change of height on the curved surface of the microscope slide may cause the samples exceeding the DOF. Thus, it needs to adjust the distance between the lens and the microscope slide manually. Therefore, the efficiency at high magnification is very low, which hence brings great obstacles to scientific research and technology development because there are many samples placed on one microscope slide and the distance needs to he readjusted during each observation of the samples.
In a previously applied patent with a publication number of CN104730702, a shooting method of a microscopic scanning platform for realizing continuous scanning is disclosed. An adjustment of the distance between the lens and the standard microscope slide on an object stage is corrected by measuring the height change of the object stage. However, the influence of the height change of the curved surface of the microscope slide on the image shot by the microscope is still not overcome.
In an invention patent with a publication number of CN102298206, a focusing method (referring to paragraph 0019 of the description) is disclosed for calculating a characteristic quantity representing a focus shift degree of an illuminated field diaphragm based on an output signal generated by an image pickup element, and for driving an illuminated field diaphragm focus adjustment mechanism based on the calculated characteristic quantity to adjust an imaging position of the illuminated field diaphragm imaging. In this invention, a thickness change (i.e., the height change of the curved surface) of the microscope slide is calculated according to difference images between two pictures shot at parts of the microscope slide with different thickness and according to an intensity difference between an output signal of a first image and an output signal of a second image. A position control part moves a position of a microscope stage toward an optical illumination system according to the thickness change calculated by a thickness change calculation module. The above solution provides a focusing method for calculating the characteristic quantity representing the focus shift degree of the illuminated field diaphragm based on the output signal generated by the image pickup element, and for driving the illuminated field diaphragm focus adjustment mechanism based on the calculated characteristic quantity to adjust the imaging position of illuminated field diaphragm imaging. However actually, it is a method with a large error when applied to a high-power microscope with magnification greater than 100 times and is unable to achieve auto-focused fast photographing and scanning.
Therefore, a method capable of accurately measuring and calculating the heights on the curved surface of the microscope slide at high magnification is provided in this disclosure, and the method is applied to the technical field of fast scanning of microscopes.